Microarray technology is a significant tool presently being used to promote progress in research in numerous fields including genomics and proteomics. This technology has broad applications to life science research, pharmaceutical and biotechnology R & D, and molecular and clinical diagnostics. Hybridization reactions between nucleic acids (or other biological moieties) are fundamental to microarray applications. These in vitro reactions will usually transpire between biological probes (oligonucleotide, cDNA, RNA, PNA, peptide, protein, etc.) bound to a solid support and free target (oligonucleotide, cDNA, RNA, PNA, peptide, protein, etc.) in solution. The probes and targets, regardless of their nature, are complementary and specific to each other. For example, for an oligonucleotide single strand probe, its target is the complementary single strand sequence. For a protein array, the target can be a protein (antigen) and its probe the target-specific antibody. Nucleic acid based microarrays are also capable of detecting specific mistakes in complementary sequences, such that a single base mismatch will significantly lower hybridization efficiency.
Hybridization of microarrays may be carried out under static conditions, without any external agitation of the hybridization target solution. Under these conditions, diffusion is limited to convection and is influenced by kinetic properties of the target (size, mobility, solution temperature) and viscosity of hybridization solution. In general, hybridization kinetics under static conditions are slow, and the resulting hybridizations become time-consuming and unpredictable. Diffusion in this case is not an entirely reliable process, which may result in decreased sensitivity and specificity of the array. Further inconsistencies in the microarray hybridization process may result from variations in array heating and orientation.
The coverslip method is generally always used with static conditions because the capillary action created by the coverslip prevents any convective solution motion. This method is preferred when the amount of target solution is limited. It involves placing a few microlitters of highly concentrated target solution onto a microarray and placing a glass or plastic coverslip directly on top of the target. The target solution then spreads into a thin layer, via capillary action, between the coverslip and the slide. Such restricted space, as available between the coverslip and slide, allows limited if any fluid movement in the film layer itself. In addition, evaporation of target solution has been known to occur, resulting in drying and precipitation of target onto the slide, and this can cause further inconsistency as well as scanning artifacts. For most dependable and consistent microarray measurements, environmental conditions and temperatures must be very strictly controlled during hybridization.
When large target volumes are available, hybridization can be carried out using mailers, staining jars, or even conical centrifuge tubes. Efficient agitation of the liquid volumes in these containers can be accomplished by rocking, shaking, etc. Properly performed, this causes thorough movement of the target solution across the microarray and results in uniform hybridization across the surface of the slide(s). Though hybridization in mailers is usually efficient and consistent when done properly, the method requires the consumption of a large volume of (perhaps expensive) target solution.
Gasket-based hybridization chamber experiments are typically carried out with a relatively small target solution volume (50-800 μl). One shortcoming of this type of hybridization chamber has been that agitation of the target solution via movement of the slide and hybridization chamber (rotation, shaking, etc.) is often insufficient to counteract the force of capillary action inherent in these hybridization chambers; therefore, sufficient mixing is often not achieved so as to produce consistency of hybridization throughout the microarray. A method for improving agitation within the chamber utilizes injection of an air bubble into the target solution, see e.g. U.S. Pat. No. 6,613,529 (Sep. 2, 2003); subsequent movement of the slide and chamber during hybridization then causes the solution to be displaced by the movement of the bubble to effect better mixing throughout the hybridization chamber. Although such a bubble mechanism provides internal mixing, unfortunately such mixing is very often not uniform across the surface of the slide. When the device is attached to a shaker (vortexer), the bubble may get trapped at one end of the chamber. Devices attached to rockers or orbital rotators (where the slide moves in a windmill like motion) may also experience problems with uniformity. In a rocker, a bubble travels up and down the surface of the slide carrying the microarray but generally follows one particular path; in an orbital rotator, the bubble moves along the inner edge of the hybridization chamber, again often following one particular path and not mixing the solution efficiently in the center region of the slide. Attempts to overcome such difficulties are described in U.S. Pat. No. 6,485,918 and in patent application Publications Ser. Nos. 2002/192,701 and 2003/87,292.
A final method to actively agitate a hybridization solution in such a reaction is via the use of automated hybridization stations. The design, capacity, and agitation mechanisms of the various commercial offerings vary. However, such hybridization stations typically cost $30,000 to $60,000, which is often cost prohibitive.
These problems are felt to be even more problematic in hybridization of 3-dimensional (3D) microarrays compared to two-dimensional (2D) microarrays; probes in such 3D microarrays are immobilized within a three-dimensional hydrogel polymer droplet (90-98% solvent), which in turn is attached to a solid support. Typically the support is a chemically functionalized glass microscope slide, though it could be any other type of solid or semi-permeable material, e.g. plastic, silicon, membrane, or metal. The number of probe-containing spots can range anywhere from 1 to 10,000. The plurality of probe spots which constitute the microarray are then exposed to target material diluted in liquid buffer to detect for hybridization. During hybridization, the target must diffuse to and into each spot to reach its complementary probe. Even for 2D arrays, the target has to be delivered to the location of the probe on the surface, and non-binding target needs to be carried away from all non-complementary probes. Therefore, adequate agitation of the target solution is critical to the efficiency and consistency of microarray hybridization reactions. Experimental conditions including temperature of hybridization, target and probe concentrations, and the rate of target delivery to and from the immobilized probes are also important. This last factor is greatly influenced by the degree to which the target solution is mixed during the hybridization reaction. Solutions that are well mixed yield consistent hybridization results, while solutions that are poorly mixed tend to be irreproducible as well as possibly having artifacts introduced.
After hybridization is complete, microarrays usually undergo a wash step; then they are dried and are scanned using a data collection device. These devices are generally confocal laser scanners, CCD (charge coupled device) cameras systems or fluorescent microscopes. The scanner emits a monochromatic light beam, which excites fluorophores bound to the microarray. The resultant emission is then filtered, collected by a photomultiplier tube (PMT), and converted to numerical intensity values. The greater the signal intensity, the greater the degree of hybridization for that particular probe/target system. Often, microarray results can be negatively influenced or even ruined by streaks, splotches, or high background on the microarray. These artifacts are typically caused by inadequate blocking prior to hybridization, inadequate solution mixing during hybridization or improper washing after hybridization.
To obviate the above-mentioned difficulties, the search has continued for improved hybridization devices.